Our lab has studied the role of protein kinase C (PKC) isozymes in cardiac ischemia for almost 20 years, using unique tools that we have developed and continue to develop. The work was initially triggered by our hypothesis that anchoring of activated PKC to subcellular sites via binding to select protein substrates, RACKs, is critical for PKC function. To prove the original RACK hypothesis, we generated new tools, short peptides that interfere with protein-protein interactions within PKC and between PKC and RACKs. These highly selective first generation peptide regulators of PKC have been instrumental in elucidating opposing (yin/yang) roles for two PKC isozymes, delta and epsilon PKC, in the responses to cardiac injury by ischemia and reperfusion (I/R). Here, we plan to expand this work while maintaining the focus on delta and epsilon PKC in models of myocardial infarction (MI). These studies will be carried out at six different `levels': in vivo, ex vivo, at the cellular level, at the subcellular, the molecular and the sub- molecular levels. Specifically, in AIM 1, we plan to identify selective substrates of each isozyme in each subcellular fraction. In AIM 2, we plan to determine the molecular basis for the opposing roles of delta and epsilon PKC during I/R. In AIM 3, we plan to use novel (second generation) PKC regulating peptides to selectively inhibit I/R-induced interaction of delta and epsilon PKC with distinct protein substrates and not with others at each particular subcellular site. The proposed study will provide a better understanding of how activation of these two homologous isozymes during I/R exerts opposing effects on outcome;I/R- induced delta PKC activation leads to cardiac infarction and activation of epsilon PKC to cardioprotection. Clearly, complex responses are elicited to both protect the cells from ischemic injury as well as to induce cell death. Our studies will continue to elucidate these processes using unique pharmacological tools that we have designed - short peptides that regulate isozyme-specific functions, selectively. Current treatment for acute myocardial infarction (AMI) is aimed at inducing reperfusion by either mechanical means (balloon catheters) or by the use of enzymatic means (thrombolytics) to disrupt the occlusion. While these treatments are effective in limiting the duration of ischemia, no therapeutic treatment is currently available to prevent ischemic injury and to reduce reperfusion injury (I/R injury) associated with these interventions. Our proposed studies will continue to elucidate these fundamentally important processes and our long term goal is that this work will identify new and highly specific therapeutics to treat patients subjected to cardiac ischemia and reperfusion. PUBLIC HEALTH RELEVANCE: Current treatment for acute myocardial infarction (AMI) is aimed at inducing reperfusion by either mechanical means (balloon catheters) or by the use of enzymatic means (thrombolytics) to disrupt the occlusion. While these treatments are effective in limiting the duration of ischemia, no therapeutic treatment is currently available to prevent ischemic injury and to reduce reperfusion injury associated with these interventions. Our proposed studies will continue to elucidate these fundamentally important processes and our long term goal is that this work will identify new and highly specific therapeutics to treat patients subjected to cardiac ischemia and reperfusion.